We have been examining how sympathetic neurons chose the neurotransmitters that they will use and how target tissues acquire the appropriate complement of receptors and effector proteins. Descriptive studies delineated a developmental change in transmitter phenotype from noradrenergic to cholinergic in the sympathetic innervation of rodent sweat glands. Transplantation and culture experiments indicate that interactions with the target tissue, mediated by a secreted differentiation factor, induce this change. Studies are in progress to identify the sweat gland factor. In one series of experiments we examined the possibility that neurotrophin-3 induces cholinergic propeties in rat sympathetic neurons are reported for chick. In contrast to the studies reported for chick, we did not find any evidence of cholinergic properties following neurotrophin-3 treatment. We have undertaken the biochemical purification of the sweat gland factor using a neuroblastoma cell line as a rapid bioassay. Significant purification has been achieved and we anticipate having enough protein to obtain sequence soon. We identified a second target of cholinergic sympathetic neurons, the periosteum. Analysis of the transmitter properties of the developing periosteal innervation coupled with transplantation studies indicate that these sympathetic neurons, like those that innervate sweat glands, undergo a target-directed transmitter switch. Like the sweat gland factor, the periosteal cholinergic factor is a member of the neuropoietic cytokine family. Sympathetic innervation of sweat glands is required for the glands to undergo a final maturation and become competent to secrete sweat in response to a cholinergic stimulus. Both adrenergic cholinergic stimulation is necessary. Studies using RT-PCR demonstrate that all five muscarinic cholinergic receptor subtypes are present in footpads but only m1 and m3 are required for secretory responsiveness. In the absence of m1 or m3, there is a significant decrease in the number of glands that acquire normal responsiveness while in the absence of both the glands do not secrete sweat. We are also examining the trophic factor responsiveness of developing sympathetic neurons in vivo. All postmitotic sympathetic neurons require NGF for survival while half also require NT-3. Both NGF and NT-3 activate the tyrosine kinase receptor, trkA.